Development Of A Cytokine Chip

细胞因子具有多种调节功能，其中最重要的是对t细胞分化的调节。分泌的细胞因子谱已被证明是预测不同的t细胞分化，特别是细胞因子调节细胞介导和体液免疫。为了在单个样品中同时测量不同的细胞因子谱，基于对感兴趣的分析物的固相免疫亲和提取，开发了一种基本的细胞因子芯片。通过羰基二咪唑桥接将重组链亲和素点阵列衍生到硅化玻璃载玻片表面。抗细胞因子抗体通过其碳水化合物部分被生物素化，并与阶梯亲和素结合。样品在分析前用激光染料标记，并用芯片孵育。通过洗涤去除非反应性物质后，结合的分析物在扫描激光密度计中读取。在固定化学和保持抗体分子活性的技术上的改进使芯片对所有50种细胞因子的灵敏度提高到1.0-2.5 pg/ml。研究人员正在继续提高这一水平的灵敏度，并进一步优化芯片读取机制。当使用细胞因子模型混合物和人类临床样品时，该芯片表现出非常好的选择性和高度的特异性。该芯片目前正处于评估阶段，然后将其用于分析重要的调节途径，包括造血、炎症神经发生调节、细胞调节和伤口愈合。

Cytokines serve a variety of regulatory functions, one of the most important being the regulation of T-cell differentiation. Secreted cytokine profiles have been demonstrated to be predictive of different T-cell differentiation, especially cytokines regulating cell-mediated and humoral immunity. In order to measure different cytokine profiles simultaneously in individual samples, a basic cytokine chip has been developed based on solid-phase immunoaffinity extraction of the analytes of interest. Arrays of recombinant streptavidin spots were derivatized to the surfaces of silanized glass microscope slides via a carbonyldiimidazole bridge. Anti-cytokine antibodies were biotinylated via their carbohydrate moieties and bound to the steptavidin. Samples were labeled with a laser dye prior to analysis and incubated with the chip. Following removal of non-reactive materials by washing, the bound analytes were read in a scanning laser densitometer. Improvements in both the immobilization chemistry and techniques for keeping the antibody molecules active have increased the sensitivity of the chip to 1.0-2.5 pg/ml for all 50 cytokines studied. Studies are continuing to improve this level of sensitivity as well as further optimize the chip reading mechanism. The chip exhibits very good selectivity and a high degree of specificity when using both model mixtures of cytokines and human clinical samples. The chip is now in the evaluation stage prior to its use in such applications as the analysis of important regulatory pathways, including hematopoesis, inflammation neurogenic regulation, cellular regulation, and wound healing. Work has continued to develop and improve a static antibody array for measuring cytokines and growth factors in clinical samples. The acquisition of an automated chip reader greatly improved both sensitivity and selectivity of the antibody arrays. Additionally, the use of a dual laser system has expanded the usefulness of the array. Collaborative studies recently established with the Critical Care Center of the NIH Clinical Center have opened up new clinical applications for the antibody chip. At present, a panel of 50-100 specific antibodies is being immunochemically evaluated for inclusion in a patient-screening chip. This chip will be used to screen pediatric patients with inflammatory lung disease for regulatory cytokines, pain-associated neuropeptides, and other modulators associated with immune function in respiratory disease.